Using dreadd for neuronal modulation in treating neuronal diseases

ABSTRACT

A method for treating a patient suffering from a neuronal hypo-kinetic disease or a neuronal hyper-kinetic disease by modulating neuronal activity in the: internal globus pallidus (GPi), in the anterior motor thalamus and/or in the external globus pallidum (GPe) and/or in the subthalamic nucleus (STN) by utilizing suppressor and/or enhancer DREADDs is provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is Continuation of U.S. patent application Ser. No.16/078,491, filed Aug. 21, 2018, which is a National Phase of PCT PatentApplication No. PCT/IL2017/050294 having International filing date ofMar. 8, 2017, which claims the benefit of priority of U.S. PatentApplication No. 62/305,601 filed on Mar. 9, 2016. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

FIELD OF THE INVENTION

A method for treating a neuronal hypo-kinetic disease or a neuronalhyper-kinetic disease by utilizing suppressor and/or enhancer DREADDs isprovided.

BACKGROUND OF THE INVENTION

Several novel strategies using engineered receptors activated bysynthetic ligands or by light have ushered in a new era of brainresearch that allows for precise experimental manipulation of neuronalactivity. These techniques are now being used to probe the involvementof discrete brain circuits in complex behaviors (Ferguson and Neumaier,2012).

One such approach uses designer receptors exclusively activated bydesigner drugs (DREADDs) to modulate cellular functions (Rogan and Roth,2011). This family of evolved muscarinic receptors has been shown toincrease (Gs-DREADD; Gq-DREADD) or decrease (Gi/o-DREADD) cellularactivity following administration of an otherwise inert syntheticligand, clozapine-n-oxide (Armbruster et al, 2007). When packaged intoviral vectors or expressed in transgenic mouse models, these tools allowcellular activity to be controlled in a defined spatial and temporalmanner. For example, activation of hippocampal neurons by Gq-DREADDreceptors amplifies γ-rhythms and increases locomotor activity andstereotypy in mice (Alexander et al, 2009). Activity of non-neuronalcells can also be controlled by DREADD receptors, as expression andactivation of either Gs-DREADD or Gq-DREADD receptors in pancreaticβ-cells increases insulin release, and repeated activation of thesereceptors leads to β-cell hypertrophy (Guettier et al, 2009).

DREADDs are mutant muscarinic receptors. A: DREADDs are formed by pointmutations in the third and fifth transmembrane regions of muscarinicreceptors (Y149C and A239G in hM3). In addition, the Gs-coupled DREADDcontains the second and third intracellular loops of the β1-AR in placeof those of the M3 muscarinic receptor. B: in human pulmonary arterysmooth muscle cells, the hM3Dq receptor (hM3D) is selectively activatedby CNO but not by ACh, resulting in PIP2 hydrolysis. Conversely, thewild-type M3 muscarinic receptor (hM3) is potently activated by ACh butnot by CNO (Armbruster et al., 2007).

DREADD receptor technology was used in a cell-specific manner to unravelthe role of striatal circuits in neuropsychiatric disorders, such asdrug addiction and obsessive-compulsive disorder. Viral vectors that useneuropeptide promoters (dynorphin or enkephalin) were used to targetDREADD receptor expression to specific cell populations in the striatum(striatonigral versus striatopallidal neurons, respectively). Someresults indicated that transiently decreasing activity ofstriatopallidal neurons in rats during repeated amphetamine exposurefacilitated the development of behavioral sensitization, whereasdisrupting activity of striatonigral neurons impaired the persistence ofthis phenomenon (Ferguson et al, 2011). Thus, these findings clearlydemonstrate that striatonigral and striatopallidal neurons havecritical, yet opposing, roles in the regulation of drugexperience-dependent behavioral plasticity.

DREADDs have also been used to control glial cell activity to modulatethe autonomic nervous system (Agulhon et al., 2013). In periphery,DREADDs have been used to control GPCR signaling in pancreaticbeta-cells (Guettier et al., 2009), hepatocytes (Li et al., 2013), andbreast cancer cells (Yagi et al., 2011).

A hypo-kinetic disorder or hypokinesia refers to decreased bodilymovement. Hypokinesia is characterized by a partial or complete loss ofmuscle movement due to a disruption in the basal ganglia. Patients withhypokinetic disorders like Parkinson's disease (PD) experience musclerigidity and an inability to produce movement. It is also associatedwith mental health disorders and prolonged inactivity due to illness,amongst other diseases.

A hyper-kinetic disorder or hyperkinesias (or hyperkinesis), refers toan increase in muscular activity that can result in excessive abnormalmovements, excessive normal movements, or a combination of both.Hyperkinesia is a state of excessive restlessness which is featured in alarge variety of disorders that affect the ability to control motormovement, such as Huntington's disease. Many hyperkinetic movements arethe result of improper regulation of the basal ganglia-thalamocorticalcircuitry. In many instances hyperkinesia is paired with hypotonia, adecrease in muscle tone. Many hyperkinetic disorders are psychologicalin nature and are typically prominent in childhood.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a method for treating asubject afflicted with a neuronal hypo-kinetic disease or disorder,comprising: suppressing neuronal activity in the internal globuspallidus (GPi); and enhancing neuronal activity in the anterior motorthalamus, the subthalamic nucleus (STN) or the external globus pallidus(GPe), wherein suppressing neuronal activity comprises transfecting GPineurons with an inhibitory DREADD and activating the inhibitory DREADD,wherein enhancing neuronal activity comprises transfecting neurons inthe anterior motor thalamus, in the subthalamic nucleus (STN) or theexternal globus pallidus (GPe), with and excitatory DREADD andactivating the excitatory DREADD, thereby treating a subject afflictedwith a neuronal hypo-kinetic disease or disorder.

In another embodiment, transfecting GPi neurons with an inhibitoryDREADD is injecting AAV viral vector comprising the Gi DREAD gene.

In another embodiment, transfecting neurons in the anterior motorthalamus, in the subthalamic nucleus (STN) or the external globuspallidus (GPe), with an excitatory DREADD is injecting AAV viral vectorcomprising the: Gq DREAD gene, Gs DREAD gene, or both.

In another embodiment, suppressing neuronal activity in the internalglobus pallidus (GPi) and enhancing neuronal activity in the anteriormotor thalamus, in the subthalamic nucleus (STN) or the external globuspallidus (GPe) are preformed concomitantly.

In another embodiment, this invention further provides that activatinginhibitory DREADD, activating excitatory DREADD, or both is contactingGPi neurons, anterior motor thalamus neurons, STN neuron, the externalglobus pallidus (GPe) or any combination thereof with CNO. In anotherembodiment, a hypo-kinetic disease or disorder is Parkinson's disease(PD).

In another embodiment, this invention further provides a method fortreating a subject afflicted with a neuronal hyper-kinetic disease ordisorder, comprising: enhancing neuronal activity in the internal globuspallidus (GPi); and suppressing neuronal activity in the anterior motorthalamus, in the subthalamic nucleus (STN) or the external globuspallidus (GPe), wherein enhancing neuronal activity comprisestransfecting GPi neurons with an excitatory DREADD and activatingexcitatory DREADD, wherein suppressing neuronal activity comprisestransfecting neurons in the anterior motor thalamus in the subthalamicnucleus (STN) or the external globus pallidus (GPe), with an inhibitoryDREADD and activating inhibitory DREADD, thereby treating a subjectafflicted with a neuronal hyper-kinetic disease or disorder.

In another embodiment, transfecting GPi neurons with an excitatoryDREADD is injecting AAV viral vector comprising the: Gq DREAD gene, GsDREAD gene, or both. In another embodiment, transfecting neurons in theanterior motor thalamus, in the subthalamic nucleus (STN) or theexternal globus pallidus (GPe) with an inhibitory DREADD is injectingAAV viral vector comprising the Gi DREAD gene or transfecting neurons inthe GPi with an excitatory DREADD (Gq) is injecting AAV viral vector.

In another embodiment, a neuronal hyper-kinetic disease or disorder ischorea, dystonia, tick-disorder, Tourette syndrome, obsessive compulsivedisorder, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

FIGS. 1A-1D are bar graphs showing the effect of CNO on normal controlmice (Cont) and mice with 6OHDA induced hemi PD (6OHDA). The effect ofblinded CNO administration was compared to NS on three behavioralparameters: the number and percent of clockwise rotations in a 5-minutemotor activity test (FIG. 1A and FIG. 1B); the mean velocity in a motoractivity test (FIG. 1C); and average time mice remained on the rotatingrod in the rotarod test (FIG. 1D). CNO had no significant effect on anyof these behavioral parameters.

FIGS. 2A-2D are bar graphs showing the effect of Unilateral Gi DREADDsin the GPi (EP) and SNr—effect of CNO on mice with 6OHDA induced hemi PDand expressing the Gi DREADD in the EP and SNr nuclei. The effect ofblinded CNO administration was compared to NS on three behavioralparameters: the number and percent of clockwise rotations in a 5-minutemotor activity test (FIG. 2A and FIG. 2B); the mean velocity in a motoractivity test (FIG. 2C); and the average time mice remained on therotating rod in the rotarod test (FIG. 2D). The beneficial effect of CNOon all behavioral parameters examined. ** p<0.01.

FIGS. 3A-3B are bar graphs showing the effect of bilateral Gi DREADDs inthe GPi (EP) and SNr—the effect of CNO on 6OHDA induced bilateral PDexpressing the Gi DREADD in both EP and SNr nuclei. The effect ofblinded CNO administration was compared to NS on two behavioralparameters: The mean velocity in a motor activity test (FIG. 3A); andthe average time mice remained on the rotating rod in the rotarod test(FIG. 3B). The beneficial effect of CNO on both the mean velocity androtarod test. ** p<0.01.

FIGS. 4A-4D are bar graphs showing the effect of unilateral Gq DREADDsin the GPe—the effect of CNO on 6OHDA induced hemi PD expressing the GqDREADD in the GPe nucleus. The effect of blinded CNO administration wascompared to NS on three behavioral parameters: the number and percent ofclockwise rotations in a 5-minute motor activity test (FIG. 4A and FIG.4B); the mean velocity in the motor activity test (FIG. 4C); and averagetime mice remained on the rotating rod in the rotarod test (FIG. 4D).The beneficial effect of CNO on all behavioral parameters examined. **p<0.01.

FIGS. 5A-5B are bar graphs showing the effect of bilateral Gq DREADDs inthe GPe—the effect of CNO on 6OHDA induced bilateral PD expressing theGq DREADD in both Gpe nuclei. The effect of blinded CNO administrationwas compared to NS on two behavioral parameters: The mean velocity in amotor activity test (FIG. 5A); and the average time mice remained on therotating rod in the rotarod test (FIG. 5B). The beneficial effect of CNOon both the mean velocity and rotarod test. ** p<0.01.

FIGS. 6A-6D are bar graphs showing the effect of unilateral Gq DREADDsin the STN—the effect of CNO on 6OHDA induced hemi PD expressing the GqDREADD in the STN nucleus. The effect of blinded CNO administration wascompared to NS on three behavioral parameters: the number and percent ofclockwise rotations in a 5-minute motor activity test (FIG. 6A and FIG.6B); the mean velocity in the motor activity test (FIG. 6C); and averagetime mice remained on the rotating rod in the rotarod test (FIG. 6D).Note the beneficial effect of CNO on turns and mean velocity, but not inthe rotarod test. ** p<0.01.

FIGS. 7A-7B are bar graphs showing the effect of bilateral Gq DREADDs inthe STN—the effect of CNO on 6OHDA induced bilateral PD expressing theGq DREADD in both STN nuclei. The effect of blinded CNO administrationwas compared to NS on two behavioral parameters: The mean velocity in amotor activity test (FIG. 7A); and the average time mice remained on therotating rod in the rotarod test (FIG. 7B). The beneficial effect of CNOon both the mean velocity and rotarod test. ** p<0.01.

FIGS. 8A-8C are bar graphs summarizing the effect of CNO on mice with6OHDA induced experimental PD expressing DREADDs in different nuclei: Giin the EP and SNr nuclei, Gq in the GPe nucleus, Gq in the STN, Gq inboth STN nuclei (upper 2 panels), Gi in the STN, Gq in the ventralthalamus, control mice and mice with 6OHDA induced hemi PD. The relativeeffect of CNO and NS (CNO/NS) is presented for three behavioralparameters: The mean velocity in a motor activity test (FIG. 8A); thetime mice remained on the rotating rod in the rotarod test (FIG. 8B);and the reduction in the percent of clockwise rotations in a 5-minutemotor activity test (FIG. 8C). ** p<0.01.

FIGS. 9A-9D are bar graphs showing unilateral Gi DREADDs in the GPi (EP)and SNr: Continues 5 day activation—the suppression of the indirectpathway by a 5-day activation of Gq DREADDs in the GPe of 6OHDA inducedPD mice. Open field and Rota-Rod tests were performed during 5-dayapplication of normal saline (NS) and CNO in 6-OHDA treated PD miceexpressing the Gq DREADDs in the GPe nucleus unilaterally. Average(mean±SEM) of the movement velocity (FIG. 9A), Rota-Rod score (FIG. 9B),number (FIG. 9C) and percent (FIG. 9D) of clockwise rotations in NS andCNO treated unilateral PD mice (n=6). ** p<0.01.

FIGS. 10A-10D are bar graphs showing unilateral Gq DREADDs in the GPe:Continues 5-day activation—a 5-day suppression of the activity of theoutput basal ganglia nuclei in 6OHDA induced PD mice. Open field andRota-Rod tests were performed during application of normal saline (NS)and CNO in 6-OHDA treated PD mice expressing the Gi DREADDs in the GPiand SNR nuclei unilaterally. Average (mean±SEM) of the movement velocity(FIG. 10A), Rota-Rod score (FIG. 10B), number (FIG. 10C) and percent(FIG. 10D) of clockwise rotations in NS and CNO treated unilateral PDmice (n=6). ** p<0.01.

FIGS. 11A-11D are bar graphs showing a combined unilateral Gi DREADDs inthe EP (GPi) and SNr and Gq DREADDs in the GPe and SNr: continuous—a5-day combined manipulation 3 targets within the basal ganglia nuclei in6OHDA induced PD mice. Open field and Rota-Rod tests were performedduring Gi DREADDs in the GPi and SNR nuclei, Gq DREADDs in the GPenucleus and a 5-day application of normal saline (NS) and CNO in 6-OHDAtreated PD mice expressing the Gq DREADDs in the STN nucleusunilaterally. Average (mean±SEM) of the movement velocity (FIG. 11A),Rota-Rod score (FIG. 11B), number (FIG. 11C) and percent (FIG. 11D) ofclockwise rotations in NS and CNO treated unilateral PD mice (n=6). **p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in one embodiment, a method formodulating bodily movement in a subject in need thereof by affectingneuronal activity in the internal globus pallidus (GPi), the externalglobus pallidus (GPe), the anterior motor thalamus, the subthalamicnucleus (STN), or any combination thereof. In one embodiment, provided amethod for improving the motor performance and/or function in a subjectafflicted with a neurological or a CNS disease which limits a motorfunction. In one embodiment, provided a method for improving the motorperformance and/or function in a subject afflicted with a neurologicalor a CNS disease which limits a motor function by targeting andmodulating the indirect pathway anatomically (GPe nucleus).

In one embodiment, enhancing STN activity by Gq DREADDs results inrestoring and/or significantly improving motor performance of motoractivities in a subject afflicted with a neuronal hypo-kinetic diseaseas demonstrated in both Hemi- and bilateral experimental PD. In oneembodiment, it was unexpectedly found that activation of both GPe andSTN yielded the same behavioral outcome, as the GPe inhibits the STN. Inone embodiment, it was unexpectedly found that activation of both GPeand STN yielded significant improvement of motor performance in asubject afflicted with a neuronal hypo-kinetic disease such as but notlimited to PD. In one embodiment, it was unexpectedly found that DREADDsmodulation of three targets within the basal ganglia complex, theindirect pathway nucleus GPe, the output nuclei GPi and SNr and the STNresulted in significant improvement of motor performance in a subjectafflicted with a neuronal hypo-kinetic disease such as but not limitedto PD.

In one embodiment, provided herein a method for significantly improvingmotor performance in a subject afflicted with a neuronal hypo-kineticdisease (such as PD) via DREADDs modulation of three targets within thebasal ganglia complex, the indirect pathway nucleus GPe, the outputnuclei GPi and SNr and the STN.

In another embodiment, the term “modulating” is altering. In anotherembodiment, the term “modulating” is activating, enhancing, restoring,ameliorating or any combination thereof. In another embodiment, the term“modulating” is inhibiting. In another embodiment, the term “modulating”is increasing. In another embodiment, the term “modulating” is inducing.In another embodiment, the term “modulating” is elevating. In anotherembodiment, the term “modulating” is reducing. In another embodiment,the term “modulating” is differentially activating. In anotherembodiment, the term “modulating” is decreasing. In another embodiment,the term “modulating” is differentially inhibiting.

In another embodiment, “modulating bodily movement” is modulating thefrequency of at least one movement. In another embodiment, “modulatingbodily movement” is modulating the amplitude of at least one movement.

In one embodiment, the present invention provides a method for treatinga subject afflicted with a neuronal hypo-kinetic disease or disorder,comprising: a. suppressing neuronal activity in the internal globuspallidus (GPi); and b. enhancing neuronal activity in the anterior motorthalamus, the external globus pallidus (GPe) the subthalamic nucleus(STN), wherein suppressing neuronal activity comprises transfecting GPineurons with an inhibitory DREADD and activating inhibitory DREADD,wherein enhancing neuronal activity comprises transfecting neurons inthe anterior motor thalamus, the external globus pallidus (GPe) or inthe subthalamic nucleus (STN) with and excitatory DREADD and activatingexcitatory DREADD, thereby treating a subject afflicted with a neuronalhypo-kinetic disease or disorder.

In another embodiment, DREADD, inhibitory DREADD, excitatory DREADD orany combination thereof is: hM3Dq coupled to Gaq (Gq) signaling andinduces firing of neurons; hM4Di coupled to Gai signaling and mediatesneuronal and synaptic silencing; and rM3Ds coupled to Gas signaling andwhich modulates neuronal activity. In another embodiment, inhibitoryDREADD is hM4Di, which coupled to Gai (Gi) signaling and mediatesneuronal and synaptic silencing. In another embodiment, excitatoryDREADD is hM3Dq, coupled to Gaq (Gs) signaling. In another embodiment,excitatory DREADD is rM3Ds, which is coupled to Gas signaling.

In another embodiment, DREADD as described herein is carried by a vectorfor express in a neuronal target tissue or cells. In another embodiment,DREADD as described herein is carried by a viral vector for express in aneuronal target tissue or cells. In another embodiment, DREADD carriedby a viral vector is sufficiently expressed in a neuronal target tissueor cells within 4 to 31 days. In another embodiment, DREADD carried by aviral vector is sufficiently expressed in a neuronal target tissue orcells within 7 to 25 days. In another embodiment, DREADD carried by aviral vector is sufficiently expressed in a neuronal target tissue orcells within 10 to 25 days. In another embodiment, DREADD carried by aviral vector is sufficiently expressed in a neuronal target tissue orcells within 7 to 21 days.

In another embodiment, any DREADD as described herein is activated byCNO. In another embodiment, any DREADD as described herein is activatedby CNO administered via parenteral administration. In anotherembodiment, any DREADD as described herein is activated by CNOadministered via oral administration.

In another embodiment, DREADDs are activated by clozapine-N-oxide (CNO).In another embodiment, are activated by clozapine-N-oxide (CNO) at adosage of between 0.1 to 20 mg/kg. In another embodiment, DREADDs areactivated by clozapine-N-oxide (CNO) at a dosage of between 1 to 5mg/kg.

In another embodiment, the present invention provides that transfectinga GPi neuron with an inhibitory DREADD is contacting a Gi DREAD genewith a GPi neuron. In another embodiment, the present invention providesthat transfecting GPi neurons with an inhibitory DREADD is injecting AAVviral vector comprising the Gi DREAD gene. In another embodiment, thepresent invention provides that transfecting neurons in the anteriormotor thalamus and/or in the subthalamic nucleus (STN) and/or theexternal globus pallidus (GPe) with an excitatory DREADD includeinjecting AAV viral vector comprising the: Gq DREAD gene, Gs DREAD gene,or both.

In another embodiment, the present invention provides that transfectingneurons in the anterior motor thalamus with an excitatory DREADD includecontacting AAV viral vector comprising the: Gq DREAD gene, Gs DREADgene, or both with neurons in the anterior motor thalamus. In anotherembodiment, the present invention provides that transfecting neurons inthe subthalamic nucleus with an excitatory DREADD include contacting AAVviral vector comprising the: Gq DREAD gene, Gs DREAD gene, or both withneurons in the subthalamic nucleus. In another embodiment, the presentinvention provides that transfecting neurons in the GPe with anexcitatory DREADD include contacting AAV viral vector comprising the: GqDREAD gene, Gs DREAD gene, or both with neurons in the Gpe. In anotherembodiment, contacting AAV viral vector comprises injecting AAV viralvector to the neuronal site as described herein. In another embodiment,contacting AAV viral vector comprises injecting AAV viral vector intoneurons as described herein.

In another embodiment, the present invention provides that suppressingneuronal activity in the internal globus pallidus (GPi) and enhancingneuronal activity in the anterior motor thalamus, and/or in the externalglobus pallidum (GPe), and/or in the subthalamic nucleus (STN) arepreformed concomitantly. In another embodiment, the present inventionprovides that CNO administration suppresses neuronal activity in theinternal globus pallidus (GPi) and enhances neuronal activity in theanterior motor thalamus and/or in the subthalamic nucleus (STN), and/orin the external globus pallidum (GPe) at once and/or concomitantly. Inanother embodiment, the present invention provides that activating aninhibitory DREADD and activating an excitatory DREADD is achieved by asingle ligand such as but not limited to CNO. In another embodiment, thepresent invention provides that activating an inhibitory DREADD isachieved by a first ligand and activating an excitatory DREADD isachieved by a second ligand. In another embodiment, the first ligand andthe second ligand do not cross react.

In another embodiment, activating the inhibitory DREADD, activating theexcitatory DREADD, or both is contacting GPi neurons expressing DREADD,anterior motor thalamus neurons expressing DREADD, STN neuron expressingDREADD, GPe neurons expressing DREADDs or any combination thereof withCNO.

In one embodiment, enhancing STN activity by Gq DREADDs results inrestoring and/or significantly improving motor performance of both Hemi-and bilateral experimental PD. In one embodiment, it was unexpectedlyfound that activation of both GPe and STN yielded the same behavioraloutcome, as the GPe inhibits the STN. In one embodiment, it wasunexpectedly found that activation of both GPe and STN yieldedsignificant improvement of motor performance in PD. In one embodiment,it was unexpectedly found that DREADDs modulation of three targetswithin the basal ganglia complex, the indirect pathway nucleus GPe, theoutput nuclei GPi and SNr and the STN resulted in significantimprovement of motor performance PD mice.

In one embodiment, DREADDs modulation of: the indirect pathway nucleusGPe, the output nuclei GPi and SNr and the STN improves and/or restoresmotor function in a subject afflicted with a disease such as described

In another embodiment, treating a subject afflicted with hypo-kineticdisease or disorder is improving motor activities in the subject. Inanother embodiment, a hypo-kinetic disease is a cardiovascular disease.In another embodiment, a hypo-kinetic disease is a form of cancer. Inanother embodiment, a hypo-kinetic disease is associated with back pain.In another embodiment, a hypo-kinetic disease is associated withdisability arising from the back. In another embodiment, a hypo-kineticdisease is obesity. In another embodiment, a hypo-kinetic disease istype 2 diabetes. In another embodiment, a hypo-kinetic disease isosteoporosis. In another embodiment, a hypo-kinetic disease isosteoarthritis. In another embodiment, a hypo-kinetic disease isassociated with a mental disease. In another embodiment, a hypo-kineticdisease is high Blood pressure.

In another embodiment, a subject afflicted with hypo-kinetic disease isafflicted with hypokinesia or decreased bodily movement. In anotherembodiment, a subject afflicted with hypo-kinetic disease is sufferingfrom damage to the basal ganglia.

In another embodiment, a subject afflicted with hypo-kinetic disease issuffering from a partial loss of muscle movement due to a disruption inthe basal ganglia. In another embodiment, a subject afflicted withhypo-kinetic disease is suffering from a complete loss of musclemovement due to a disruption in the basal ganglia. In anotherembodiment, a subject afflicted with hypo-kinetic disease is afflictedwith Parkinson's disease (PD). In another embodiment, a subjectafflicted with hypo-kinetic disease experiences muscle rigidity and aninability to produce movement.

In another embodiment, a subject afflicted with hypo-kinetic disease issuffering from akinesia or a severe case of Parkinson's disease. Inanother embodiment, a subject afflicted with hypo-kinetic disease issuffering from bradykinesia or “stone face” (expressionless face). Inanother embodiment, a subject afflicted with hypo-kinetic disease issuffering from dysarthria. In another embodiment, a subject afflictedwith hypo-kinetic disease is suffering from dyskinesia. In anotherembodiment, a subject afflicted with hypo-kinetic disease is sufferingfrom dystonia. In another embodiment, a subject afflicted withhypo-kinetic disease is suffering from freezing characterized by aninability to move muscles in any desired direction. In anotherembodiment, a subject afflicted with hypo-kinetic disease is sufferingfrom neuroleptic malignant syndrome. In another embodiment, a subjectafflicted with hypo-kinetic disease is suffering from supranuclearpalsy. In another embodiment, a subject afflicted with hypo-kineticdisease is suffering from an increase in muscle tone. In anotherembodiment, a subject afflicted with hypo-kinetic disease is sufferingfrom ‘Cogwheel’ rigidity. In another embodiment, a subject afflictedwith hypo-kinetic disease is suffering from ‘leadpipe’ rigidity. Inanother embodiment, a subject afflicted with hypo-kinetic disease issuffering from postural instability.

In another embodiment, “treating” is reducing muscle rigidity. Inanother embodiment, “treating” is reducing muscle rigidity. In anotherembodiment, “treating” is increasing the range of motion of at least onelimb. In another embodiment, “treating” is increasing the range ofmotion of at least one organ. In another embodiment, “treating” isalleviating symptoms associated with a hypo-kinetic disease or disorder.

In one embodiment, DREADD is used to modulate the activity of a nucleuswithin the cortico-basal ganglia loop. In one embodiment, DREADD is usedto counterbalance network abnormalities caused by a disease as describedherein (such as but not limited to PD). In one embodiment, DREADD isused for increasing motor activity is subjects afflicted with a diseaseas described herein. In one embodiment, DREADD is used for reducingbasal ganglia output activity.

In one embodiment, DREADD is a Gq DREADD. In one embodiment, DREADD is aGi DREADD. In one embodiment, a nucleus is the external globus pallidum(GPe) nucleus. In one embodiment, a nucleus is the subthalamic nucleus(STN). In one embodiment, a nucleus is internal globus pallidum (GPi).

In another embodiment, “treating” is reducing alterations of cerebralcirculation. In another embodiment, “treating” is reducing blood flow inthe supramarginal gyms and angular gyms of the parietal lobe. In anotherembodiment, “treating” is reducing cardiac activity and changes in thetonus of the heart vessels. In another embodiment, “treating” isalleviating non-motor symptoms associated with Parkinson's disease.

In another embodiment, “treating” is alleviating neuropsychiatricdisturbances. In another embodiment, “treating” is alleviating cognitivedisturbances. In another embodiment, “treating” is improvingvisuospatial difficulties. In another embodiment, “treating” is reducingthe risk of dementia. In another embodiment, “treating” is reducing thefrequency and/or severity of behavior and mood alterations. In anotherembodiment, “treating” is alleviating depression. In another embodiment,“treating” is alleviating apathy. In another embodiment, “treating” isalleviating anxiety. In another embodiment, “treating” is reducing therisk of psychotic symptoms. In another embodiment, “treating” isreducing the risk of hallucinations or delusions. In another embodiment,“treating” is alleviating the sleep impairment. In another embodiment,“treating” is reducing the risk of orthostatic hypotension. In anotherembodiment, “treating” is reducing the risk of oily skin. In anotherembodiment, “treating” is reducing the risk of excessive sweating. Inanother embodiment, “treating” is reducing the risk of urinaryincontinence. In another embodiment, “treating” is reducing the risk ofsexual dysfunction. In another embodiment, “treating” is reducing therisk of gastric dysmotility. In another embodiment, “treating” isreducing the risk of eye and vision abnormalities such as decreasedblink rate, dry eyes, deficient ocular pursuit (eye tracking) andsaccadic movements (fast automatic movements of both eyes in the samedirection). In another embodiment, “treating” is reducing difficultiesin directing gaze upward. In another embodiment, “treating” is reducingthe risk of blurred or double vision. [In another embodiment, “treating”is reducing the risk of impaired sense of smell. In another embodiment,“treating” is reducing the risk of sensation of pain. In anotherembodiment, “treating” is reducing the risk of paresthesia (skintingling and numbness). In another embodiment, “treating” is reducingside effects associated with permanent damage to the brain. In anotherembodiment, “treating” is free of causing permanent damage to the brainby the treatment of the invention.

In another embodiment, enhancing neuronal activity is increasingneuronal frequency. In another embodiment, enhancing neuronal activityis increasing neuronal input, output or both. In another embodiment,enhancing neuronal activity is increasing an action potential. Inanother embodiment, enhancing neuronal activity is enhancing the rate atwhich neurons fire. In another embodiment, enhancing neuronal activityis increasing the activity of a neuron. In another embodiment, enhancingneuronal activity is inducing the activity of a neuron. In anotherembodiment, enhancing neuronal activity is generating oscillatoryactivity. In another embodiment, neuronal activity is measured by anymethod or means known to one of skill in the art.

In another embodiment, suppressing neuronal activity is decreasingneuronal frequency. In another embodiment, suppressing neuronal activityis decreasing neuronal input, output or both. In another embodiment,suppressing neuronal activity is decreasing an action potential. Inanother embodiment, suppressing neuronal activity is decreasing the rateat which neurons fire. In another embodiment, suppressing neuronalactivity is decreasing the activity of a neuron. In another embodiment,suppressing neuronal activity is inhibiting oscillatory activity.

In another embodiment, the present invention provides a method fortreating a subject afflicted with a neuronal hyper-kinetic disease ordisorder, comprising: a. enhancing neuronal activity in the internalglobus pallidus (GPi); and b. suppressing neuronal activity in theanterior motor thalamus or the external globus pallidum (GPe) or in thesubthalamic nucleus (STN), wherein enhancing neuronal activity comprisestransfecting GPi neurons with an excitatory DREADD and activating theexcitatory DREADD, wherein suppressing neuronal activity comprisestransfecting neurons in the anterior motor thalamus or in thesubthalamic nucleus (STN), or in the external globus pallidum (GPe) withan inhibitory DREADD and activating the inhibitory DREADD, therebytreating a subject afflicted with a neuronal hyper-kinetic disease ordisorder.

In another embodiment, transfecting GPi neurons with an excitatoryDREADD is injecting AAV viral vector comprising the: Gq DREAD gene, GsDREAD gene, or both. In another embodiment, transfecting neurons in theanterior motor thalamus or the external globus pallidum (GPe) or in thesubthalamic nucleus (STN) with an inhibitory DREADD is injecting AAVviral vector comprising the Gi DREAD gene. In another embodiment,enhancing neuronal activity in the internal globus pallidus (GPi) andsuppressing neuronal activity in the anterior motor thalamus or theexternal globus pallidum (GPe) or in the subthalamic nucleus (STN) areperformed at once and/or concomitantly. In another embodiment,activating inhibitory DREADD and activating excitatory DREADD isachieved by a single ligand such as but not limited to CNO.

In another embodiment, treating a subject afflicted with a neuronalhyper-kinetic disease or disorder or a neuronal hypo-kinetic disease ordisorder is improving motor activities in the subject. In anotherembodiment, treating a subject afflicted with a neuronal hyper-kineticdisease or disorder is without inducing permanent damage to the brain.

In another embodiment, a hyper-kinetic disease or disorder ishyperkinesias or hyperkinesis. In another embodiment, a hyper-kineticdisease is Huntington's disease. In another embodiment, a hyper-kineticdisease further comprises hypotonia. In another embodiment, ahyper-kinetic disease or disorder is chorea, dystonia, tick-disorder,Tourette syndrome, hemi balism, or any combination thereof. In anotherembodiment, a hyper-kinetic disease or disorder is athetosis. In anotherembodiment, a hyper-kinetic disease or disorder is an ataxia. In anotherembodiment, a hyper-kinetic disease or disorder is Hemiballismus.

In another embodiment, a hyper-kinetic disease or disorder is Tardivedyskinesia. In another embodiment, a hyper-kinetic disease or disorderincludes stereotypies. In another embodiment, a hyper-kinetic disease ordisorder includes myoclonus. In another embodiment, a hyper-kineticdisease or disorder includes hemifacial spasm. In another embodiment, ahyper-kinetic disease or disorder includes tardive dystonia. In anotherembodiment, a hyper-kinetic disease or disorder is Wilson's disease. Inanother embodiment, a hyper-kinetic disease or disorder includesvolitional hyperkinesias. In another embodiment, a hyper-kinetic diseaseor disorder includes tremor. In another embodiment, a hyper-kineticdisease or disorder includes restless leg syndrome.

In another embodiment, a hyper-kinetic disease or disorder includespost-stroke repercussions.

In another embodiment, a hyper-kinetic disease or disorder includesdentatorubral-pallidoluysian Atrophy.

In one embodiment, a subject afflicted with a neuronal hypo-kineticdisease such as PD is treated according to the methods described hereinby modifying the activity of different nuclei in the basal ganglia loop.In one embodiment, altering the activity of different nuclei in thebasal ganglia loop is achieved by using Designer Receptors ExclusivelyActivated by Designer Drugs (DREADD) (example 2). In one embodiment,provided herein a method for reducing the output of the inhibitory GPiand SNr nuclei to the ventral thalamus. In one embodiment, providedherein a method for increasing the excitatory drive to the neocortex.

In one embodiment, provided herein a method for improving the motorsymptoms of PD by targeting three different nuclei in the basal ganglialoop: the GPi and SNr nuclei, which serve as the output nuclei of thebasal ganglia loop; the GPe, which is exclusively involved in theindirect pathway; and the STN, which serves as the main target for deepbrain stimulation (DBS) in PD patients. In one embodiment, the inventionprovides the inhibition of neuronal firing in inhibitory output nuclei(the GPi and SNr nuclei). In one embodiment, the invention provides theinhibition of neuronal firing in inhibitory output nuclei (the GPi andSNr nuclei) via Gi DREADDS. In one embodiment, the invention providesthe activation of Gi expressed in the STN. In one embodiment, treatingPD or inhibiting a side effect associated with PD comprises theactivation of Gq DREADDS expressed in the STN. In one embodiment,treating PD or inhibiting a side effect associated with PD comprises thebilateral Gq activation. In one embodiment, treating is increasing,restoring or enhancing a motor activity.

In one embodiment, a “physiologically acceptable carrier” and/or a“pharmaceutically acceptable carrier” are combined with a DREADDactivator such as CNO. In one embodiment, the phrases “physiologicallyacceptable carrier” and “pharmaceutically acceptable carrier” which beinterchangeably used refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered modulator. Anadjuvant is included under these phrases. In one embodiment, one of theingredients included in the pharmaceutically acceptable carrier can befor example polyethylene glycol (PEG), a biocompatible polymer with awide range of solubility in both organic and aqueous media (Mutter etal. (1979)).

In one embodiment, “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA,latest edition, which is incorporated herein by reference.

In one embodiment, suitable routes of administration, for example,include oral, rectal, transmucosal, transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,emulsions and the like. Such unit dosage forms comprise a safe andeffective amount of the desired modulator, or modulators. Thepharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.

In some embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and croscarmelose; lubricants such as magnesium stearate,stearic acid and talc. In one embodiment, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjutants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets.

In some embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in some embodiments areaqueous solutions or emulsions comprising a safe and effective amount ofthe modulator of the present invention and optionally, other compounds.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intramuscular injection of a liquidpreparation. In some embodiments, liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment, the pharmaceutical compositions are administeredintravenously, and are thus formulated in a form suitable forintravenous administration. In another embodiment, the pharmaceuticalcompositions are administered intra-arterially, and are thus formulatedin a form suitable for intra-arterial administration. In anotherembodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In another embodiment, a modulator is delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the modulator. In oneembodiment, the pack, for example, comprise metal or plastic foil, suchas a blister pack. In one embodiment, the pack or dispenser device isaccompanied by instructions for administration. In one embodiment, thepack or dispenser is accommodated by a U.S. Food and Drug Administrationfor prescription drugs or of an approved product notice associated withthe container in a form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals, which notice isreflective of approval by the agency of the form of the compositions orhuman or veterinary administration. Such notice, in one embodiment, islabeling approved by the insert.

In one embodiment, it will be appreciated that the modulator of thepresent invention can be provided to the individual with additionalactive agents to achieve an improved therapeutic effect as compared totreatment with each agent by itself. In another embodiment, measures(e.g., dosing and selection of the complementary agent) are taken toadverse side effects which are associated with combination therapies.

EXAMPLES Material and Methods Animals

Adult C57 black mice are used the current experiments.

Vector

DREADDs are provided within viral particles. Viral particles injectedin-situ.

Two viral vectors are used AAV-hSyn-hM3D(Gq)-mCherry and theAAV-hSyn-hM4D(Gi)-mCherry. The viruses are injected with a stereotacticapparatus to the targeted brain region (either GPi, thalamus, GPe orsubthalamic nucleus).

Parkinson Model

The 6OHDA model was used.

Behavioral Test

To monitor the motor impairment and the ability of the DREADD therapy toreverse these impairments the following behavioral tests are conducted.Open field test: to measure motor activity dysfunction. In this test thespontaneous movement of mice in a square arena are recorded andquantified. Rotarod test: Mice were placed on a rotating rod withrotation speed gradually increasing from 4 to 40 RPM using a presetacceleration program. The time mice fell from the rotating rod wasmeasured, with 300 seconds being the maximal time measured on the rod.

Example 1: DREADDs Impact in Hyperkinesia and Hypokinesia

C57 black mice are injected with the viral vector (eitherAAV-hSyn-hM3D(Gq)-mCherry which contains the gene for the excitatoryDREADD Gq or AAV-hSyn-hM4D(Gi)-mCherry which contains the gene for theinhibitory DREADD (Gi) is injected into the targeted brain regions(either GPi, GPe, STN or anterior thalamus alone or in combination).Injections are performed using a micro-injector mounted on astereotactic apparatus. After viral intraperitoneal injections 6OHDA isadministered (streotactic injections) to generate the experimentalParkinsonian mice.

Approximately 3 weeks after induction of experimental Parkinson'sdisease by 6OHDA, behavioral experiments are performed. The DREADDmolecule (either Gq, Gi or both) is activated by IP injection of CNO. Inthese experiments, the behaviors of Parkinsonian mice under controlconditions (no injections), injection of CNO and sham injection ofsaline are compared.

The motor performance of mice is monitored in the open field test, theRotarod test.

The following DREADD containing viral vector injections are performed:

1) The Gi DREADD is injected into the GPi bilaterally. 2) The Gq DREADDare injected into the thalamus bilaterally. 3) Either the Gi or the GqDREADD are injected into the STN bilaterally. 4). The Gq DREADD isinjected into the GPe bilaterally.

Moreover, viral vectors containing DREADDs are simultaneously injectedinto two separate brain regions to obtain synergistic effects: 1) The GiDREADD is injected into both the GPi and the STN bilaterally. 2) The GiDREADD is injected into the GPi and the Gq DREADD are injected into theSTN bilaterally. 3) The Gi DREADD is injected into the GPi and the GqDREADD are injected into the thalamus bilaterally. 4). The Gq DREADD isinjected into the GPe bilaterally.

Example 2: Chemogenetic Treatment of Parkinson's Disease

This set of experiments provide evidence that network abnormalitiesassociated with PD can be corrected by modifying the activity ofdifferent nuclei in the basal ganglia loop using Designer ReceptorsExclusively Activated by Designer Drugs (DREADD) as describe herein.

Experiments were performed on 2-4 month old wild type C57 black mice. Toinduce experimental PD, 1 microliter of a solution containing 6 hydroxydopamine (6-OHDA) (3 mg/1 ml) was injected into the medial forebrainbundle via a small craniotomy drilled in the skull. The mouse head wasfixated in a stereotaxic frame, and the 6OHDA containing solution wasinjected using a glass pipette held by a micro-manipulator and amicro-injector.

Mice were anesthetized throughout the procedure with isoflurane. The6OHDA was injected into the MFB unilaterally or bilaterally to inducebilateral experimental PD.

Shortly after the 6OHDA injection, after recuperation from anesthesia,mice started to rotate in the clockwise direction (ipsilateral to theinjection side). Mice were treated with IP glucose and normal saline inthe first few days after the 6OHDA injection. In addition, mice had freeaccess to a sucrose solution for the first week after injection.Behavioral experiments were performed at least 3 weeks after theinduction of experimental PD.

DREADD Expression:

To express DREADDs (either the inhibitory DREADD Gi or the excitatoryDREADD Gq) viral vectors were injected (eitherAAV8-hSyn-hM3D(Gq)-mCherry or AAV8-hSyn-hM4D(Gi)-mCherry). The viralvectors were injected through a craniotomy with a glass pipette held bya micromanipulator and a micro-injector. During the injections the headwas fixated in a stereotaxic frame for accurate injections. Mice wereanesthetized throughout the procedure with isoflurane.

The viral vectors (200-50011.1) were injected into several targets inthe basal ganglia loop including the STN (either Gi and Gq), the EP andSNr nuclei (Gi), GPe (Gq) and the ventral thalamus (Gq). The injectionswere usually unilateral at the side of the 6OHDA injections. However, incases of bilateral 6OHDA injections viral vectors were injected to thetarget nuclei bilaterally. Usually viral vectors and 6OHDA were injectedat the same session.

To confirm the location of DREADD expression, at the end of allexperiments the brain was removed and fixated in paraformaldehyde (4%).Few days later the brain was sectioned (100 μm axial sections) with avibrotome, and DREADD expression was imaged by fluorescent imaging ofthe fluorescent protein mCherry. Anatomical location of DREADDexpression was determined by concomitant bright field imaging of brainsections.

Administration of Clozapine-N-Oxide (CNO) and Normal Saline

To investigate the effect of DREADD activation, 50011.1 of either IP CNO(5 mg/kg) or normal saline (NS-0.9% NaCl) was administered.Administration of either CNO or saline was performed in a blindedmanner. The vials containing the CNO and NS were label with a numericalcode by a second investigator that did not administer the IP dose, noranalyzed behavior. Behavioral experiments were performed approximately20-30 minutes after IP drug administration.

Behavioral Tests

Two behavioral tests were used for monitoring the behavior of mice: (1)Open field test: Mice were placed in a 30 cm×30 cm×30 cm open top boxfor 5 minutes and continuous recorded on a video. The results wereanalyzed off-line after the experiments ended. Using the EthoVisionsoftware, the mean velocity and distance traveled by the mice weremonitored during the 5 minutes of open field test. In addition, thenumber and direction of 180° turns were monitored with the EthoVisionsoftware. Open field tests were performed on two consecutive days (20-30minutes after mice received the blinded drug (either NS or CNO)). (2)Rotarod test: Mice were placed on a rotating rod with rotation speedgradually increasing from 4 to 40 RPM using a preset accelerationprogram. The time mice fell from the rotating rod was measured, with 300seconds being the maximal time measured on the rod. For each testingsession, the rotarod test was performed on 3 consecutive days. Duringthe first day mice underwent 4 training sessions. On the remaining 2days, mice were tested once a day (20-30 minutes after they received theIP drug (either CNO or NS in a blinded manner)).

The results provided below show improvement of PD motor symptomsobtained by targeting three different nuclei in the basal ganglia loop:the EP (GPi equivalent in primates) and SNr nuclei, which serve as theoutput nuclei of the basal ganglia loop; the GPe, which is exclusivelyinvolved in the indirect pathway; and the STN, which serves as the maintarget for deep brain stimulation (DBS) in PD patients.

The Entopeduncular Nucleus (EP-Rodent GPi) and the Pars Reticulata ofthe Substantia Nigra

The EP and SNr nuclei serve as the output nuclei of the basal ganglialoop, where the direct, indirect and hyper-direct pathways converge. Theaim of this experiment was to treat PD or PD symptoms by inhibitingfiring in the SNr and EP nuclei (the rodent equivalent of the primateGPi) using the Gi DREADDS.

Specifically, blinded IP administration of normal saline (NS) and CNOwere compared in both hemi-parkinsonian (6-OHDA injected to the MFB) andcontrol mice. The behavioral parameters that were examined includedipsilateral turning and movement velocity in the open field test andtime to falling off the rotating rod in the rotarod test.

In normal control and mice with experimental 6OHDA induced hemi PD nosignificant differences between blinded IP administration of CNO and NSwere observed on any of the three behavioral parameters we examined(FIG. 1 ).

In contrast, in mice with 6OHDA induced hemi PD mice and expressing GiDREADDS in the EP and SNr nuclei blinded CNO application resulted insignificantly and surprisingly better behavioral performances comparedto NS in all behavioral parameters examined. As compared to NS, CNOcaused a 36±9% improvement in the mean velocity in the open field test;a 68.9±10.8% reduction in the number and 46±8.6% reduction in thepercent of clockwise rotations; and a 30.5±7.1% increase in the timespent on the rotating rod in the rotarod test (FIG. 2 ).

Targeting the External Globus Pallidum Nucleus (GPe)

In this set of experiments, the GPe nucleus, which serves as a majorrelay nucleus exclusively belonging to the indirect pathway, wastargeted. More specifically, the GPe neurons were activated using theexcitatory Gq DREADDs, and the effect on behavior on 6-OHDA inducedhemi-parkinsonian mice, was monitored.

These experiments showed that blinded administration of CNO resulted ina significant improvement of all behavioral parameters as compared tonormal saline (NS) interperitoneally (IP) administered. CNOadministration resulted in an increase of 77.2±24.7% in the meanvelocity in the open field test; a 37.7±20% reduction in the number and% reduction in the percent of clockwise rotations; and a 25.6±6.1%increase in the time spent on the rotating rod in the rotarod test (FIG.4 ). Thus suppressing the activity of the indirect pathway byDREADD-mediated activation of the GPe nucleus significantly improvedperformances of all motor parameters examined in both unilateral andbilateral 6-OHDA-induced PD mice. The effects of DREADD-mediatedactivation of the GPe on motor performance and/or function were morepronounced in the case of bilateral 6-OHDA induced PD (Gq DREADDs wereexpressed uni- and bilaterally in the GPe of hemi- and bilateralparkinsonian mice, respectively).

The Sub Thalamic Nucleus (STN)

In contrast to the GPe nucleus, STN neurons participate in both theindirect and hyper direct pathways. The effect of both inhibitory orexcitatory DREADDs expressed in the STN on behavior of experimental6OHDA induced PD was tested.

When the effect of blinded IP administration of CNO to NS was comparedon the open field and rotarod tests, it was found that activation of Giexpressed in the STN did not significantly affect the mean velocity norperformances in the rotarod test. Activation of Gi DREADDs expressed inthe STN nucleus did show a small, yet significant beneficial effect onturns.

In contrast, activation of Gq DREADDS expressed in the STN had large,unexpected and significant beneficial effects on 6-OHDA experimental PD.CNO administration resulted in an increase of 117.6±26.9% in the meanvelocity in the open field test; a 37.2±17% reduction in the number and47.6±11.1% reduction in the percent of clockwise rotations. Theperformances in the rotarod test showed no significant differencesbetween blinded CNO and NS application in this group (FIG. 6 ).

In addition, the effect of bilateral Gq activation on bilateralexperimental PD (6OHDA injected to the MFB bilaterally), was tested. Itwas found that Gq activation in these mice had a dramatic unexpectedeffect. CNO administration resulted in a 294.4±70.9% increase in themean velocity in the open field test and an 81±15.8% increase in thetime before falling off the rotarod (FIG. 7 ). In this case turns werenot analyzed as to begin with mice with bilateral experimental PD showedlittle tendency to rotate to either side (0.1±0.06 turns per minute).

FIG. 8 presents a comparison between the different DREADD mediatedmanipulations that were performed in these experiments.

Combined Suppression of the Output Nuclei and Indirect Pathway

The results obtained in this section further support the findingsprovided hereinabove. In this set of experiments the NS and CNO wereapplied intraperitoneally daily for 5 consecutive days. The open fieldtest was performed on day 2 and 4 (for both CNO and NS groups). Rota-Rodtest was performed on days 3 and 5. The results of the motor velocity,rotations and Rota-Rod scores were compared between the normal salineand CNO application.

The following conditions were examined. Further support for the impactof suppression of the output nuclei: activation of Gi via DREADDs in theGPi and SNR nuclei in hemi-parkinsonian mice is provided in FIG. 9 .Further support for the impact of suppression of the indirect pathway onmotor activity by Activation of Gq DREADDs in the GPe nucleus inhemi-parkinsonian mice is provided in FIG. 10 . Evidence for the impactof combined suppression of the output nuclei and indirect pathway andactivation of the STN via simultaneous Activation of Gi DREADDs in theGPi and SNR nuclei, of Gq DREADDs in the GPe nucleus and of Gq DREADDsin the STN is provided in FIG. 11 .

These sets of experiments demonstrate that targeting different nucleiand pathways within the basal ganglia complex in experimentalParkinson's disease result in unexpected improved motor performanceand/or function of experimental PD. Specifically, suppressing theactivity of the indirect pathway by targeting the GPe nucleus;activating the STN; and suppressing the output activity of the basalganglia by targeting the output nuclei the GPi and SNr resulted in anunprecedented restoration and/or improvement of motor performance and/orfunction in mice afflicted with PD.

What is claimed is:
 1. A method for treating a subject afflicted with aneuronal hyper-kinetic disease or disorder, comprising: (a) enhancingneuronal activity in the internal globus pallidus (GPi); and (b)suppressing neuronal activity in the: anterior motor thalamus, externalglobus pallidum (GPe), the subthalamic nucleus (STN), or any combinationthereof, wherein said enhancing neuronal activity comprises transfectingsaid GPi neuron with an excitatory DREADD and activating said excitatoryDREADD, wherein said suppressing neuronal activity comprisestransfecting a neuron in: the anterior motor thalamus, the externalglobus pallidum (GPe), or the subthalamic nucleus (STN) with aninhibitory DREADD and activating said inhibitory DREADD, therebytreating a subject afflicted with a neuronal hyper-kinetic disease ordisorder.
 2. The method of claim 1, wherein said transfecting said GPineuron with an excitatory DREADD is injecting AAV viral vectorcomprising the: Gq DREAD gene, Gs DREAD gene, or both.
 3. The method ofclaim 1, wherein said transfecting a neuron in: the anterior motorthalamus, the external globus pallidum (GPe), or the subthalamic nucleus(STN) with an inhibitory DREADD is injecting AAV viral vector comprisingthe Gi DREAD gene.
 4. The method of claim 1, wherein said enhancingneuronal activity in the internal globus pallidus (GPi) and saidsuppressing neuronal activity in: the anterior motor thalamus, theexternal globus pallidum (GPe), or the subthalamic nucleus (STN) arepreformed concomitantly.
 5. The method of claim 1, wherein saidactivating said inhibitory DREADD and activating said excitatory DREADDis achieved by a single ligand.
 6. The method of claim 1, wherein saidactivating said inhibitory DREADD, activating said excitatory DREADD, orboth is contacting: a GPi neuron, an anterior motor thalamus neuron, aGPe neuron, a STN neuron or any combination thereof with CNO.
 7. Themethod of claim 1, wherein said treating a subject afflicted with aneuronal hyper-kinetic disease or disorder is improving motor activitiesin said subject.
 8. The method of claim 1, wherein said treating asubject afflicted with a neuronal hyper-kinetic disease or disorder isalleviating non-motor symptoms.
 9. The method of claim 1, wherein saidtreating a subject afflicted with a neuronal hyper-kinetic disease ordisorder is without inducing permanent damage to the brain.
 10. Themethod of claim 1, wherein said neuronal hyper-kinetic disease ordisorder is chorea, dystonia, tick-disorder, Tourette syndrome, or anycombination thereof.